"Reperfusion" refers to the re-establishment of blood flow to a cell, a tissue or an organ after a period of time in which blood flow to the same collection of cells, tissue or organ was partially or completely interrupted, i.e., "ischemia." Reperfusion also refers to the flow of "normoxic" blood, i.e., blood in which the oxygen content is sufficient to satisfy normal cellular oxygen requirements, to a collection of cells, a tissue or an organ after a period of time in which "hypoxic" blood, i.e., blood in which the oxygen content is insufficient to satisfy normal cellular oxygen requirements, was flowing to the same collection of cells, tissue or organ.
Reperfusion is often associated with a number of diseases and conditions/events as well as a number of medical procedures. It is most commonly associated with stroke, heart attack, cardiac surgery, in particular cardiac surgery involving cardiopulmonary bypass, and transplantation of cells, a tissue or an organ.
Reperfusion often results in injury--thus, the term "reperfusion injury." Reperfusion injury can run the gamut from subcellular damage to gross cellular damage. For example, reperfusion can result in the disruption of nucleic acid sequencing, the destruction of cell membrane integrity, the failure of normally tight intercellular junctions, as well as hemorrhage and edema.
The exact cascade of events initiated by reperfusion and leading to injury is unclear. It is likely that the cascade of events, which involves the release of multiple cellular products, including thromboxanes, components of complement, such as C3a and C5a, and free radicals, leads to unbridled destruction by the normally selective and contained defense system of the body. Central to this defense system is the NADPH-oxidase enzyme, which was first characterized in leukocytes but now has been identified in multiple other types of cells and tissues, including monocytes, smooth muscle cells, endothelial cells, the carotid body and lung tissue.
The NADPH-oxidase enzyme is a complex of membrane-bound and cytosolic components. The complex is unassembled, i.e., inactive, in quiescent cells. Upon cell perturbation, e.g., by infection, however, the membrane-bound and cytosolic components assemble to form the active complex. The membrane-bound component includes cytochrome b558, which consists of a 22 kD protein (p22.sub.[phox]) bound tightly to a 91 kD glycosylated integral membrane protein (gp91.sub.[phox]). The cytosolic component comprises a 47 kD protein (p46.sub.[phox]), a 67 kD protein (p67.sub.[phox]), and a low molecular weight GTP-binding protein.
NADPH-oxidase transfers electrons from NADPH to oxygen, resulting in the generation of reactive oxygen species, such as O.sub.2.sup.- and H.sub.2 O.sub.2, in an intense process referred to as "oxidative burst." Under normal conditions, the reactive oxygen species generated by NADPH-oxidase are part of the body's defense system against, for example, microorganisms. However, during reperfusion, NADPH-oxidase can play a role in cellular destruction.
Four-hydroxy-3-methoxyacetophenone, commonly referred to as "apocynin," is a known inhibitor of NADPH-oxidase. It is a compound that has been isolated from the root of the plant Picrorhiza kurroa, which grows in the Himalayan mountains. Extracts of Picrorhiza kurroa have been used in acute and chronic preparations, given topically, systemically, intravascularly or orally, to treat inflammatory diseases, arthritis, sepsis-related lung injury, liver disease, lung disease, fever, skin lesions, wound infections, rheumatic disease, urinary disorders, heart failure, and snake and scorpion bites. It has been disclosed as useful in the treatment of acute and chronic inflammations of the airways, joints and blood vessels (EP 551662). It also has been disclosed as useful in the treatment of a metabolic condition, namely atherosclerosis (WO 97/19679). It also has been suggested to be useful in the prevention of thrombosis (Engels et al., FEBS Letter 305: 254-256 (1992)).
It now has been surprisingly and unexpectedly discovered that inhibitors of NADPH-oxidase, such as apocynin, are useful in the prophylactic and therapeutic treatment of reperfusion injury. Accordingly, it is a principal object of the present invention to provide a method of prophylactically and therapeutically treating reperfusion injury, such as that resulting from ischemia or hypoxia. This and other objects of the present invention will become apparent from the detailed description provided herein.